Effect of reference conditions on flow rate, modifier fraction and retention in supercritical fluid chromatography.

When using compressible mobile phases such as fluidic CO2, the density, the volumetric flow rates and volumetric fractions are pressure dependent. The pressure and temperature definition of these volumetric parameters (referred to as the reference conditions) may alter between systems, manufacturers and operating conditions. A supercritical fluid chromatography system was modified to operate in two modes with different definition of the eluent delivery parameters, referred to as fixed and variable mode. For the variable mode, the volumetric parameters are defined with reference to the pump operating pressure and actual pump head temperature. These conditions may vary when, e.g. changing the column length, permeability, flow rate, etc. and are thus variable reference conditions. For the fixed mode, the reference conditions were set at 150bar and 30°C, resulting in a mass flow rate and mass fraction of modifier definition which is independent of the operation conditions. For the variable mode, the mass flow rate of carbon dioxide increases with system pump operating pressure, decreasing the fraction of modifier. Comparing the void times and retention factor shows that the deviation between the two modes is almost independent of modifier percentage, but depends on the operating pressure. Recalculating the set volumetric fraction of modifier to the mass fraction results in the same retention behaviour for both modes. This shows that retention in SFC can be best modelled using the mass fraction of modifier. The fixed mode also simplifies method scaling as it only requires matching average column pressure.

Understanding and diminishing the extra-column band broadening effects in supercritical fluid chromatography.

Supercritical fluid chromatography, where a low-viscosity mobile phase such as carbon dioxide is used, proves to be an excellent technique for fast and efficient separations, especially when sub-2µm particles are used. However, to achieve high velocities when using these small particles, and in order to stay within the flow rate range of current SFC-instruments, narrow columns (e.g. 2.1mm ID) must be used. Unfortunately, state-of-the-art instrumentation is limiting the full separation power of these narrower columns due to significant extra-column band broadening effects. The present work identifies and quantifies the different contributions to extra-column band broadening in SFC such as the influence of the sample solvent, injection volume, extra-column volumes and detector cell volume/design. When matching the sample solvent to the mobile phase in terms of elution strength and polarity (e.g. using hexane/ethanol/isopropanol 85/10/5vol%) and lowering the injection volume to 0.4µL, the plate count can be increased from 7600 to 21,300 for a low-retaining compound (k'=2.3) on a 2.1mm×150mm column (packed with 1.8µm particles). The application of a water/acetonitrile mixture as sample solvent was also investigated. It was found that when the volumetric ratio of water/acetonitrile was optimized, only a slightly lower plate count was measured compared to the hexane-based solvent when minimizing injection and extra-column volume. This confirms earlier results that water/acetonitrile can be used if water-soluble samples are considered or when a less volatile solvent is preferred. Minimizing the ID of the connection capillaries from 250 to 65µm, however, gives no further improvement in obtained efficiency for early-eluting compounds when a standard system configuration with optimized sample solvent was used. When switching to a state-of-the-art detector design with reduced (dispersion) volume (1.7-0.6µL), an increase in plate count is observed (from 11,000 to 14,000 plates on a 2.1mm×100mm column with 1.8µm particles for k'=3) even when 250µm tubing was used. Using this detector cell and decreasing the ID of the tubing from 250 to 120µm resulted in an additional increase to 17,300 plates. Further decreasing the tubing ID (e.g. 65µm) appeared to have no observable influence on the obtained plate count.

Exploring the speed-resolution limits of supercritical fluid chromatography at ultra-high pressures.

The limits of supercritical fluid chromatography have been experimentally explored using inlet pressures at the limits of the current commercial instrumentation (400-600 bar), as well as pressures significantly surpassing this (up to 1050 bar). It was found that efficiencies in the range of 200,000 theoretical plates can be achieved for a void time t0 of roughly 6min using superficially porous particles (2.7 and 4.6µm) while remaining within the pressure limits of current commercial instrumentation and columns. If lower efficiencies are sufficient (<100, 000 plates), smaller particles (e.g. 1.8µm) provide the best trade-off between analysis time and efficiency. Apparent efficiencies of 83,000 (k'=2.2) to 76,000 (k'=6.6) plates could be achieved for void times around 1min by pushing the pressure limits up to 1050 bar on a column length of 500mm. As the optimal mobile phase velocity for these small particle columns is even higher, it is required to use narrow-bore columns (2.1mm ID) to remain within the instrument limits of flow rate. The smaller column volume however puts a strain on the separation efficiency due to extra-column band broadening, resulting in losses up to 50% for weakly retained compounds for column lengths below 250mm. It is also illustrated that when using sub-2µm particles, especially for separations where a significant amount of organic modifier is required, the current pressure limits of state-of-the-art instrumentation can sometimes be insufficient. For a gradient running from 4 to 40 v% methanol on a 300mm column at the optimal flow rate the pressure increases from 420 to 810 bar. Operating SFC-columns with a large pressure gradient induces several (undesired) side effects which have been investigated as well. It has been found that, since the viscosity increases strongly with pressure in SFC, the optimal flow rate and the minimal plate height can significantly change when the column length is changed. Whereas e.g. a 3×150mm column (2.7µm particles) has an optimal flow rate of 1.5ml/min and minimal plate height of 5.66µm, a 3×1050mm column has an optimal flow rate of 1.2ml/min and a minimal plate height of 6.25µm. Nevertheless, an increase in operating pressure drop in SFC results in a significant gain in kinetic performance.